Project Summary Several human proteins are known to form amyloid fibrils, and these fibrils are associated with several devastating diseases, including Alzheimer?s, Parkinson?s, type II diabetes, and dialysis-related amyloidosis (DRA). One of these proteins, human ?-2- microglobulin (?2m), can form amyloid fibrils in the presence of Cu(II), leading to DRA. While many general aspects of amyloid formation are understood, molecular-level information about the early stages of amyloid forming reactions is only beginning to be revealed. This information, though, is critical for the rational development of therapeutics against amyloid diseases. Previous work from our group has developed and applied new covalent labeling (CL) techniques together with mass spectrometry (MS) to structurally characterize the pre-amyloid oligomers of ?2m. The insight gained from these new tools has helped identify small molecules that can inhibit ?2m amyloid formation. Understanding the molecular basis of these inhibitors is important for improving them, and we will advance new MS-based approaches to gain this understanding. MS is emerging as a powerful tool for studying the structures of proteins and protein oligomers, but most often individual MS-based techniques are used to gather the desired information. We propose the combination of MS-based structural tools that together will provide synergistic information to characterize the molecular basis of ?2m amyloid inhibitors. Specifically, we will explore the combination of CL/MS and hydrogen/deuterium exchange (HDX)/MS to give a more detailed picture of the structural changes undergone by ?2m and its oligomers. We will also establish an HDX-enhanced ion mobility method to characterize isomeric oligomers that are populated in the presence of certain inhibitors. Our research on ?2m amyloid formation so far has revealed how Cu(II) partially unfolds ?2m, allowing it to form distinct oligomers before mature fibrils are produced. We have also begun to study inhibitors of ?2m amyloid formation. The combination of MS-based methods proposed here seek to reveal the molecular basis of inhibitors of ?2m amyloid formation, including characterization of isomeric oligomers. A specific outcome of this research will be a better molecular-level understanding of how to inhibit ?2m amyloid formation, which will lead to therapeutics against DRA. The techniques developed in this work will be generally applicable to other amyloid systems.